A unique regulatory phase of DNA methylation in the early mammalian embryo

Abstract

DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methylcytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and so far no base-resolution maps exist to support and refine it. Here we generate genome-scale DNA methylation maps in mouse gametes and from the zygote through post-implantation. We find that the oocyte already exhibits global hypomethylation, particularly at specific families of long interspersed element 1 and long terminal repeat retroelements, which are disparately methylated between gametes and have lower methylation values in the zygote than in sperm. Surprisingly, the oocyte contributes a unique set of differentially methylated regions (DMRs)--including many CpG island promoters--that are maintained in the early embryo but are lost upon specification and absent from somatic cells. In contrast, sperm-contributed DMRs are largely intergenic and become hypermethylated after the blastocyst stage. Our data provide a genome-scale, base-resolution timeline of DNA methylation in the pre-specified embryo, when this epigenetic modification is most dynamic, before returning to the canonical somatic pattern.

Figure 1. Global CpG methylation dynamics across early murine embryogenesis

a. Schematic representation of samples isolated and purified for methylation analysis with replicate number (n) highlighted. hpf: hours post fertilization; dpf: days post fertilization. b. Fraction of 100bp tiles with High (≥0.8, red), Intermediate (Inter, >0.2 and <0.8, green) and Low (≤0.2, blue) methylation values. Brain, heart and liver tissue are shown for adult comparisons. c. Histogram of methylation values across 100bp tiles. n is the number of tiles for each stage. d. Boxplots of methylation values at different local CpG densities highlight the difference between hypomethylated pre-implantation tissues and the adult pattern seen in sperm, post-implantation and somatic samples. Bulls-eye indicates the median, edges the 25^th/75^th percentile and whiskers the 2.5^th/97.5^th percentile. e. CpG density of >0.2 methylation (left panel) and ≤0.2 methylation (right panel) tiles in stages that display somatic versus embryonic patterning (red and blue lines, respectively).

Figure 2. Major transitions in DNA methylation levels during early development

a. 100bp tiles available for pairwise comparison across consecutive embryonic stages. Tiles that remain unchanged (stable) at the indicated transitions are shown in light blue. Tiles that change by greater than 0.2 and are significant by t-test are highlighted in dark blue. b. 100bp tiles with increasing (red) or decreasing (green) methylation levels at each consecutive transition show that major transitions are largely unidirectional. c. Boxplot of methylation levels for sperm-specific DMRs (n=134,038 tiles). Red line indicates the median, edges the 25^th/75^th percentile and whiskers the 2.5^th/97.5^th percentile. d. Boxplot of methylation levels for oocyte-specific DMRs (n=6,394 tiles) as in (c). e. 74 CpGs within sperm-specific DMR tiles (c) could be ascribed to paternal and maternal alleles and tracked across stages. Paternal CpG methylation values (blue line, median; colored space, 25^th/75^th percentile) exhibit marked decrease by the zygote stage while maternal CpG methylation (red line, median; colored space, 25^th/75^th percentile) remain unchanged. If untracked, these CpGs have an intermediate methylation value between those ascribed to a parent-of-origin (black line).

Figure 3. Specific families of LINE and LTR retroelements exhibit the most dramatic changes in the sperm to zygote transition

a. Histogram of the difference in methylation levels, where negative values represent tiles decreasing from sperm to zygote, within LINE retroelement features that are captured by RRBS. 85% of the elements have a significant difference (P<0.04, FDR<0.05; t-test). The distribution is bimodal with 18% of elements displaying a change in methylation status by greater than 0.45 as highlighted in red. b. Differences in methylation between sperm and zygote within annotated LTR retroelements. Compared to LINEs, a smaller fraction of elements appear regulated by DNA demethylation (61% significant, 10% of those sampled exhibiting changes greater than 0.45 as highlighted in red). c–e. Boxplots of methylation levels in oocyte, sperm and zygote (top panels) as well as the distributions of change in methylation levels between sperm and zygote (bottom panels) for specific LINE-1 families, including those that are (c,d) or are not dynamic (e). Top panels: Red line indicates the median, edges the 25^th/75^th percentile and whiskers the 2.5^th/97.5^th percentile. Bottom panels: members of each family that are demethylated by greater than 0.45 are highlighted in red. f–h. Boxplots of methylation levels in oocyte, sperm and zygote (top panels) and the distributions of change in methylation levels between sperm and zygote (bottom panels) for specific families of LTR containing retroelements, including MMERGLN (f), RLTR10C (g) and IAP elements (h). Top and bottom panels as in (c–e). i. Mean methylation level for all elements of the L1Md_A LINE (solid blue line) and IAP LTR class (solid red line) that do not dramatically change contrasted by LINEs (dashed blue line) and LTR elements (dashed red line) that show the greatest loss at fertilization. SINE elements (green line) are less methylated in sperm than other repeat elements and are generally demethylated to oocyte levels.

Figure 4. Differentially methylated regions represent discrete gamete specific feature classes

a. Heatmap of methylation levels (black: 0; red: 1; grey: missing value) in 376 identified 100bp tiles (rows) that behave as oocyte-contributed DMRs in the zygote. Tiles are sorted by functional classes and clustered within each class. 15 known ICRs, shown at the bottom, behave similarly in the early embryo and retain intermediate methylation through implantation. b. Genomic features (top) and promoters of different CpG densities (bottom) in oocyte-contributed DMRs. Top: oocyte DMRs are enriched for promoters. Bottom: most of the 105 promoters that overlap oocyte-contributed DMR tiles are high CpG density promoters containing CpG Islands (HCPs, light blue). c. Heatmap of methylation levels (black: 0; red: 1; grey: missing value) in 4,894 identified 100bp tiles (rows) that behave as sperm-contributed DMRs in pre-implantation embryos. Tiles are sorted by functional classes (labels, left) and clustered within each class. Known DMRs contributed by sperm areat the bottom. d. Genomic features in sperm-contributed DMRs are generally intergenic.

Figure 5. DMRs resolve after cleavage to univalent hyper- or hypo- methylated values in a gamete-of-origin specific fashion

a. Single CpG resolution methylation within 2kb of the Cpne7 promoter in gametes and across embryonic development (rows). Dark gray bar highlights the CpG island. A CpG proximal to this island can be tracked to a phase resolving SNP and this region is highlighted in light gray, with paternal (X1) and maternal (C57) methylation values highlighted as an inset for each trackable phase. Values for SNP methylation in “Cleavage” correspond exactly to those captured in the zygote. Blue bars: CpG methylation; Red bars: CpA methylation. b. Composite plot of CpG (blue) and CpA (red) methylation for all HCPs (left) and for promoters that are specifically hypermethylated in oocytes (TSS DMRs, right). The region +/− 2kb of the TSS is marked in gray. Identified promoter DMRs contributed by the oocyte are hypermethylated around the periphery of the TSS and resolve to intermediate values throughout cleavage. An expected HCP methylation architecture is re-acquired for these DMRs around implantation. c. Mean methylation levels and stage for oocyte-contributed DMRs in promoters (red, dashed line) versus our complete set (red, solid line). d. Sperm-contributed DMRs (blue line) generally resolve to hypermethylation.